Trpm4 channel inhibitors for stroke treatment

ABSTRACT

The present invention relates to methods for treating ischemic stroke including extension of the therapeutic time window for reperfusion. More particularly, the invention relates to a method of treating stroke in a subject by inhibiting the transient receptor potential melastatin 4 (TRPM4) channel. The present invention also provides uses of TRPM4 inhibitors, TRPM4 antibodies and kits for use in the methods of the invention.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/392,213, filed Dec. 23, 2015, which is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/SG2014/000314, filed Jun. 30, 2014, which claims priority to Singapore Application No. 201305129-7, filed Jun. 28, 2013. The entire teaching of the above applications are incorporated herein by reference. International Application PCT/SG2014/000314 was published under PCT Article 21(2) in English.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of ischemic stroke including extension of the therapeutic time window for reperfusion. More particularly, the present invention relates to blocking the function of the TRPM4 channel to improve capillary integrity during the acute and chronic phase of stroke.

BACKGROUND OF THE INVENTION

Stroke is a major health problem worldwide. It is the 4th leading cause of death in Singapore. For those stroke patients who survive, most are likely to be left disabled and in need of significant rehabilitation. Stroke generates a greater disability impact than any other medical condition and has a huge impact on both the family and society.

There are two types of stroke: ischemic and hemorrhagic. Ischemic stroke, which accounts for more than 80% of all stroke incidences, is attributed by the atherosclerotic occlusion or embolism within an artery, commonly the middle cerebral artery. Focal ischemic stroke with sufficient severity and duration leads to infarction and persistent neurological dysfunction.

Reperfusion is the only potent therapy for acute ischemic stroke. Reperfusion aims to lyse the thrombus with recombinant tissue-type plasminogen activator (rt-PA) or other mechanical devices. Reperfusion therapy is best given within a very narrow time window (<4.5 hours after stroke onset). After that, reperfusion greatly increases cerebral edema and the risk of hemorrhagic transformation which are mainly caused by vascular damage. As very few stroke patients can arrive at hospitals and be diagnosed within this time frame, less than 5% of patients receive reperfusion therapy. Therefore, the focus of acute stroke treatment is to extend the therapeutic time window. However, numerous attempts from both pharmaceutical companies and stroke research community have failed to achieve this goal.

Transient receptor potential melastatin 4 (TRPM4) (see, for example, SEQ ID NOs: 11-13) is a voltage-dependent, non-selective monovalent cation channel. It is impermeable to Ca²⁺, activated by elevated cytosolic Ca²⁺, and modulated by ATP [Vennekens R, Nilius B., Handb Exp Pharmacol, 269-85 (2007b)]. TRPM4 belongs to the mammalian TRP superfamily. TRPM4 and TRPM5 are unique because they only conduct monovalent cations, whereas most other TRP channels are permeable to both monovalent and divalent ions. TRPM4 is important for the function of immune cells, including dendritic, mast, and T cells [e.g., Vennekens R, et al., Nat Immunol 8, 312-20 (2007a)]. When activated, TRPM4 can depolarize the membrane potential and regulate Ca²⁺ homeostasis by decreasing the driving force for Ca²⁺ entry. Gain-of-function mutations in TRPM4 are associated with familial heart disease [Kruse M, et al., J Clin Invest, 119, 2737-44 (2009)]. TRPM4 also participates in the pathophysiology of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) [Gerzanich V, et al., Nat Med, 15, 185-91 (2009); Schattling B, et al., Nat Med, 18, 1805-11 (2012)]. Ectopic expression of TRPM4 has been found in capillaries after SCI and in neurons after EAE. Activation of TRPM4 in SCI and EAE results in unchecked ion influx and subsequently leads to oncotic cell death.

Cerebral edema following brain injury is bound to cell death. Edema resulting from ischemic stroke leads to tissue damage and worsens neurological functions. Recently, upregulation of the non-selective cation channel NC_(Ca-ATP) was observed in neurovascular cells, including astrocytes, neurons, and vascular endothelia, after ischemic stroke [Simard J M, et al., Nat Med, 12, 433-40 (2006)]. Enhanced NC_(Ca-ATP) current can lead to unchecked Na⁺ entry, subsequently oncotic cell death, and is believed to cause brain edema [Kahle K T, et al., Physiology (Bethesda), 24, 257-65 (2009)]. The current exhibits many properties similar to those of TRPM4, including a smaller single-channel conductance, permeability to Na⁺ and Cs⁺, and activation by intracellular Ca²⁺ [Simard J M, et al., Nat Med, 12, 433-40 (2006)].

The NC_(Ca-ATP) current is also involved in other central nervous system injuries, including traumatic brain injury, spinal cord injury, and subarachnoid hemorrhage [Simard J M, et al., J Neurosurg, 113, 622-9 (2010)]. However, studies of NC_(Ca-ATP) channel in stroke have mainly focused on the sulfonylurea receptor-1 (SUR1), an auxiliary subunit of K_(ATP) channels [Simard J M, et al., Nat Med, 12, 433-40 (2006)]. After CNS injury, SUR1 has been found upregulated in neurons, astrocytes, and endothelial cells, and the expression is not coupled with K_(ATP) functions. There is evidence that SUR1 can associate with TRPM4, and it is believed that blocking SUR1 with a sulfonylurea such as glibenclimide could inhibit the SUR1/TRPM4 channel and salvage brain tissues after injury [Walcott B P, et al., Neurotherapeutics 9:65-72 (2012)]. However, TRPM4 homomers are not sensitive to glibenclamide and there are contradicting reports on whether SUR1 binds TRPM4 directly and whether glibenclimide has a therapeutic effect on stroke [Sala-Rabanal M, et al., J Biol Chem 287:8746-56 (2012); Woo S K, et al., J Biol Chem M112.428219 (2012); Favilla C G, et al., Stroke 42:710-5 (2011); Kunte H, et al., Ann Neurol 72:799-806 (2012)].

After the acute stage, the focus of stroke therapy is to promote angiogenesis and neurogenesis, aiming to improve functional recovery and quality of life of patients. As neurological deficits are severe and always lead to disability among stroke survivors, there is also an urgent need to improve current chronic treatment for stroke recovery.

In view of the above deficiencies, it is desirable to provide a method for extending the window for acute therapy by reducing cerebral edema, and improve current therapy for stroke recovery.

The role of TRPM4 after ischemic stroke is unclear. Rat permanent and transient middle cerebral artery occlusion models (MCAO) were used to investigate the expression and functions of TRPM4 in ischemic stroke and the possibility of inhibiting TRPM4 was tested.

SUMMARY OF THE INVENTION

The TRPM4 inhibitors identified in this study appear to have sufficient clinical efficacy for development into agents for stroke treatment.

Accordingly, in a first aspect, the present invention provides an isolated antibody or fragment thereof specific to transient receptor potential melastatin 4 (TRPM4) protein (represented by, for example, SEQ ID NOs 11-13), wherein the antibody specifically binds to a peptide sequence which lies between S5 and the P-loop of the TRPM4 protein and inhibits TRPM4 activity.

In a preferred embodiment of the invention, the antibody or fragment thereof is raised using a TRPM4 peptide selected from the group comprising a peptide consisting of the amino acid sequence SEQ ID NO: 1, a peptide consisting of the amino acid sequence SEQ ID NO: 2 and a peptide consisting of the amino acid sequence SEQ ID NO: 3, or an antigenic variant or fragment thereof.

Another preferred embodiment of the invention relates to the antibody or fragment thereof being a polyclonal, monoclonal or humanized antibody.

Another preferred embodiment of the invention relates to the antibody or fragment thereof being a mouse-human chimeric antibody.

A preferred embodiment of the invention relates to a mouse-human chimeric antibody, wherein the mouse VH domain is ligated to human IgG1 CH domain and the mouse VL domain is ligated to human light chain kappa constant (CL) domain.

In another preferred embodiment of the invention, the antibody or fragment thereof inhibits TRPM4 currents.

In another preferred embodiment of the invention, the antibody or a fragment thereof is for use in treating ischemic stroke.

According to another aspect of the invention, there is provided a method of treating ischemic stroke, comprising administering to a subject in need thereof an efficacious amount of at least one TRPM4 inhibitor.

In a preferred embodiment of the method of the invention, the at least one inhibitor is an antibody or a fragment thereof which specifically binds to TRPM4, or is a TRPM4-specific siRNA.

In another preferred embodiment of the method of the invention, the antibody is a polyclonal antibody, a monoclonal antibody, or a humanized antibody or a fragment thereof.

In another preferred embodiment of the method of the invention, the siRNA comprises, essentially consists of, or consists of a sense oligonucleotide SEQ ID NO: 7 and an antisense oligonucleotide SEQ ID NO: 8.

In another preferred embodiment of the method of the invention, the at least one TRPM4 inhibitor is administered in combination with one or more thrombolytic agents.

In another preferred embodiment of the method of the invention, the at least one TRPM4 inhibitor is administered during the acute stage and/or the chronic stage.

In another preferred embodiment of the method of the invention, the treatment increases angiogenesis in the subject.

In another preferred embodiment of the method of the invention, the treatment reduces infarct volume in the subject.

In another preferred embodiment of the method of the invention, the treatment extends the therapeutic time window for reperfusion.

In another aspect of the invention, there is provided the use of at least one TRPM4 inhibitor for the preparation of a medicament for the treatment of ischemic stroke.

In a preferred embodiment of the use of the invention, the at least one inhibitor is an antibody or a fragment thereof which specifically binds to the sequence which lies between S5 and the P-loop of TRPM4, or is a siRNA which specifically inhibits TRPM4 activity.

In another preferred embodiment of the use of the invention, the antibody is a polyclonal antibody, a monoclonal antibody, or a humanized antibody, or a fragment thereof.

In another preferred embodiment of the use of the invention, the siRNA comprises, essentially consists of, or consists of a sense oligonucleotide SEQ ID NO: 7 and an antisense oligonucleotide SEQ ID NO: 8.

In another aspect of the invention, there is provided a process for the production of an antibody or fragment thereof, comprising administering to a mammal a TRPM4 peptide, variant or antigenic fragment thereof as defined herein.

In a preferred embodiment, the peptide is selected from the group comprising a peptide consisting of the amino acid sequence SEQ ID NO: 1, a peptide consisting of the amino acid sequence SEQ ID NO: 2 and a peptide consisting of the amino acid sequence SEQ ID NO: 3, or an antigenic variant or fragment thereof.

Another aspect of the invention provides an isolated nucleic acid molecule capable of expressing the TRPM4 antigenic peptide, variant or fragment thereof according to any aspect of the present invention.

In yet another aspect of the present invention there is provided at least one plasmid or vector comprising the nucleic acid molecule according to any aspect of the present invention. Another aspect of the invention provides a kit for treating stroke, the kit comprising at least one antibody or a fragment thereof as defined herein and/or at least one siRNA as defined herein and, optionally, at least one thrombolytic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Amino acid sequence (SEQ ID NO: 11) of rat TRPM4 channel protein. Peptide sequences used to generate antibodies are highlighted and underlined.

FIG. 2: Amino acid sequence (SEQ ID NO: 12) of human Isoform 1 TRPM4b channel protein. Peptide sequences corresponding to those used to generate antibodies are highlighted and underlined.

FIG. 3: Amino acid sequence (SEQ ID NO: 13) of mouse Isoform 1 TRPM4 channel protein. Peptide sequences corresponding to those used to generate antibodies are highlighted and underlined.

FIG. 4: Schematic showing the region of the TRPM4 protein where the peptide epitope is derived from, and where antibody M4P binds to inhibit activity.

FIG. 5: Up-regulation of TRPM4 in the vascular endothelium within the penumbra region 1 day after MCAO. (A) DAB staining of the capillaries with vWF in the contralateral and ipsilateral regions. Scale bar: 200 μm. Capillary counting showed significant angiogenesis in the ipsilateral region. *** P<0.0001, n=25. (B) Representative staining of TRPM4 and co-localization with vWF in the contralateral and ipsilateral regions. Scale bar: 50 μm.

FIG. 6: (A), Immunohistochemical staining of vWF (red) and TRPM4 (green) in the sham-operated rat brain. (B), Co-staining of vWF (red) and TRPM5 (green) within penumbra region 1 day after MCAO.

FIG. 7: Time-dependent expression of TRPM4 in the endothelium within the penumbra region after MCAO. Co-staining for TRPM4 and vWF was performed at 0 hour, 6 hours, 1 day, 3 days, and 7 days post operation. Scale bar: 50 μm.

FIG. 8: The expression and function of TRPM4 in human umbilical vein endothelial cells (HUVECs). (A) RT-PCR for TRPM4 in HUVECs 1 day after OGD. GAPDH was used as a loading control. N, normoxia; H, hypoxia. After normalization to GAPDH, TRPM4 was significantly increased in HUVECs after ODG treatment. *** P<0.001, n=4. (B) Western blot for TRPM4 in HUVECs 1 day after OGD. β-actin was used as a loading control. N, normoxia; H, hypoxia. After normalization to β-actin, TRPM4 was significantly increased in HUVECs after ODG treatment. * P<0.05, n=4. (C) Immunofluorescence staining of TRPM4 (green) in HUVECs 1 day after hypoxic induction. The nuclei were labeled with DAPI (blue). Scale bar: 20 μm. (D) The effects of 5 μM of 9-phenanthrol on tube formation in HUVECs after OGD. 9-phenanthrol treatment significantly increased tube branches after OGD (n=4). N-C: normoxia control; N-P: normoxia with 9-phenanthrol; H-C: hypoxia control; H-P: hypoxia with 9-phenanthrol. * P<0.05; *** P<0.001.

FIG. 9: (A), HUVEC cell number count after treatment with different doses of 9-phenamthrol. (B), HUVEC cell number count after treatment with 5 μM 9-phenamthrol. A significant cell loss was observed after 24 hours exposure to hypoxic condition. There is no difference between control cells and 9-phenanthrol treated cells under both normoxia and hypoxia. (C), MTT assay showed no difference between cells and 9-phenanthrol treated cells, similar to cell counting study.

FIG. 10: Knockdown of TRPM4 in vivo enhances angiogenesis and reduces infarct volume after MCAO in rats. (A) Representative Western blot of TRPM4 in rat brains 1 day post MCAO. HEK 293 cells transfected with mouse TRPM4 (mTRPM4) were used as the positive controls and HEK 293 cells transfected with GFP vector alone (vc) were used as the negative control. Samples from ipsilateral penumbra region (L) and corresponding contralateral region (R) with saline (− siRNA) or siRNA treatment (+ siRNA) were probed with an antibody against TRPM4. The protein size of the rat TRPM4 (indicated by the arrow) was slightly smaller than the control mouse TRPM4. Without siRNA treatment, a two-fold increase of TRPM4 expression was found in the penumbra region (L). * P<0.05; n=4. In vivo siRNA application successfully inhibited TRPM4 up-regulation within the penumbra region. (B) Staining of the penumbra region with vWF showed the fragmentation of capillaries (indicated by the arrows) in the saline-treated rats 3 days post MCAO. Elongated intact capillaries were identified in the siRNA-treated rats. Scale bar: 50 μm. The number of capillaries was increased by two-fold with the siRNA treatment. # P<0.0001; n=7. (C) TTC staining of the rat brains 1 day post MCAO. There was a significant reduction of infarct volume by siRNA treatment. ** P<0.01; n=5.

FIG. 11: TRPM4 inhibition reduces infarction and improves motor function in a transient rat stroke model. (A) TTC staining of the rat brains 24 hours after a two-hour MCAO. siRNA against TRPM4 reduces infarction. (B). Motor functions measured by rotarod indicate an enhanced functional recovery by TRPM4 inhibition. (C). Calculation of infarct volume reveals a reduction of overall infarct volume by siRNA treatment against TRPM4. (D). Infarct reduction is more prominent at the cortex region. (E). Infarct is significantly reduced at the 5^(th) and 6^(th) of coronal sections.

FIG. 12: TRPM4 siRNA treatment successfully downregulated TRPM4 expression in a transient stroke model and improved angiogenesis. (A) Immunostaining of TRPM4 in vascular endothelium within the penumbra region in the rat brains following 2 hours MCAO. In scramble siRNA treated animals, TRPM4 colocalizes with vWF, a marker for endothelium, whereas in siRNA treated animals, TRPM4 expression is weakened in most capillaries. Scale bar 50 nm. The measurement of vascular diameter (B) and length (C) indicates pro-angiogenesis effect of siRNA. * p<0.0001; # p=0.0003.

FIG. 13: TRPM4 knockdown via siRNA improves motor function during the acute phase of stroke. (A) Rotarod performance of the sham-operated, saline-treated, and TRPM4-siRNA treated MCAO rats. The data represent the percentage of the mean duration from 3 trials at each time point after normalization to the baseline control prior to the operation. The performance of the siRNA-treated rats was better than that of the saline-treated rats at days 1, 3, and 5. siRNA vs saline: # P<0.0001; n_(sham)=5; n_(saline)=6-7; n_(siRNA)=18-21. (B) Rotarod performance at day 3 post MCAO. Both sham-operated and siRNA-treated rats performed better than the saline-treated rats. # p<0.0001. There was no difference between the sham-operated and siRNA-treated animals.

FIG. 14: Transient effect of TRPM4-siRNA treatment post stroke. (A) TTC staining at day 1 and day 5 after MCAO. (B) Infarct volume measurement at day 1, 5, and 7 after MCAO. ** P<0.01; n=5.

FIG. 15: Blocking TRPM4 with antibody reduced OGD induced cell death. (A) In HEK cells transfected with TRPM4 (green), M4P antibody (red) bind to the TRPM4 channels on the surface of live cells. Control antibody from rabbit IgG did not bind to the membrane TRPM4 channels. (B) M4P treatment significantly reduced hypoxia (12 hours incubation) induced cell death in HEK cells transfected with TRPM4. C M4P treatment significantly reduced chemicals (Sodium Azide and 2-Deoxy-D-glucose) induced cell death in HEK cells transfected with TRPM4. ** P<0.05.

FIG. 16: Under oxygen glucose deprivation (OGD) for 24 hours, M4P treatment increases cell survival both in HEK cells transfected with mouse TRPM4 (A) and in human cerebral vascular endothelial cells (B).

FIG. 17: In HEK cells transfected with mouse TRPM4, M4P is able to bind to the surface of the live HEK cells after 30 min incubation (upper row). After prolonged incubation for 2 days, M4P is internalized into cytosol of HEK cells (lower row). Therefore, M4P likely to inhibit TRPM4 in two mechanisms: by blocking TRPM4 channel in the acute stage and by downregulate surface TRPM4 expression via internalizing membrane TRPM4 protein in the chronic stage.

FIG. 18: M4P antibody at 60 μg/ml concentration is able to reduce the currents from mouse TRPM4 channels expressed in HEK cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Certain terms employed in the specification, examples and appended claims are collected here for convenience.

The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. The term “TRPM4 peptide sequence”, as used herein, refers to an antigenic peptide epitope used to generate TRPM4 inhibitory antibodies.

In this context, “fragments” refers to a TRPM4 peptide epitope according to the invention which has been reduced in length by one or more amino acids and which retains antigenic activity of TRPM4 sufficient to raise antibodies that inhibit TRPM4 activity.

The term ‘variants’ of the oligopeptide, peptide, polypeptide, or protein sequence as used herein, refers to changes that may be made to the native amino acid sequence that still allow the production of antibodies that inhibit TRPM4 activity. For example, one or more conservative amino acid substitutions may be made to the TRPM4 peptide epitope used according to the invention. Basis for this can be found in that the antibody M4P binds to the rat, mouse and human TRPM4 channels which vary in amino acids 1, 3-5, 9, 11, 13, 18, 20, 21 and 24 of the epitope peptide (Table 1). Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

An antibody is any immunoglobulin, including antibodies and fragments thereof that bind to a specific epitope. The antibody according to the invention may be prepared against the sequence which lies between S5 and the P-loop of the TRPM4 protein. More specifically, the target TRPM4 polypeptide epitope comprises, essentially consists of, or consists of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (Table 1) or a variant or fragment thereof. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab′, F(ab)′ fragments and/or F(v) portions of the whole antibody which are capable of binding to TRPM4 and inhibiting its activity. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, an expression construct such as a plasmid, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

The term “humanized antibody,” as used herein, refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

As used herein, the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope “A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

The term ‘variant’, as used in the context of the present invention is intended to describe variations to the amino acid sequence of the TRPM4 polypeptide epitope that do not remove the antigenicity of the polypeptide in terms of eliciting antibodies which bind to and inhibit TRPM4 activity. Variants include conservative amino acid substitutions.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a TRPM4 polypeptide epitope comprising, essentially consisting of, or consisting of the amino acid sequence QDRSSNCSAERGSWAHPEGPVAGSCVSQ (SEQ ID NO: 1), RDSDSNCSSEPGFWAHPPGAQAGTCVSQ (SEQ ID NO: 2) or QDRSGNCSMERGSWAHPEGPVAGSCVSQ (SEQ ID NO: 3) or a fragment thereof may be replaced with one or more other amino acid residues from the same side chain family without significantly reducing the antigenicity of the epitope or deviating significantly from the scope of the present invention.

The term “oligonucleotide,” as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay. As used herein, the term “oligonucleotide” is substantially equivalent to the terms “amplimers,” “primers,” “oligomers,” and “probes,” as these terms are commonly defined in the art.

The term “antisense,” as used herein, refers to any composition containing a nucleic acid sequence which is complementary to a specific polynucleotide sequence. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.

The term “small interfering RNA” (siRNA), as used herein, refers to small pieces of double-stranded (ds) RNA, usually about 21 nucleotides long, with 3′ overhangs (2 nucleotides) at each end that can be used to interfere with the translation of proteins by binding to and promoting the degradation of messenger RNA (mRNA) at specific sequences. In doing so, they prevent the production of specific proteins based on the nucleotide sequences of their corresponding mRNA. Suitable siRNA's for use according to the invention include SEQ ID NOs: 7-8 (Table 1).

The term “treatment”, as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.

The term “comprising” as used in the context of the invention refers to where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term “comprising” encompasses the more restrictive terms “consisting essentially of” and “consisting of.” With the term “consisting essentially of” it is understood that the epitope/antigen of the present invention “substantially” comprises the indicated sequence as “essential” element. Additional sequences may be included at the 5′ end and/or at the 3′ end. Accordingly, a polypeptide “consisting essentially of” sequence X will be novel in view of a known polypeptide accidentally comprising the sequence X. With the term “consisting of” it is understood that the polypeptide, polynucleotide and/or antigen according to the invention corresponds to at least one of the indicated sequence (for example a specific sequence indicated with a SEQ ID Number or a homologous sequence or fragment thereof).

A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.

In a first aspect, the present invention provides an isolated antibody or fragment thereof specific to transient receptor potential melastatin 4 (TRPM4) protein, wherein the antibody specifically binds to a peptide sequence which lies between S5 and the P-loop of the TRPM4 protein and inhibits TRPM4 activity.

Suitable peptide sequences (SEQ ID NOs: 1-3) are shown in Table 1.

TABLE 1  Polypeptide and polynucleotide sequences of the invention. SEQ Protein/ ID nucleotide Sequences NO Rat TRPM4  QDRSSNCSAERGSWAHPEGPVAGSCVSQ 1 epitope Human TRPM4 RDSDSNCSSEPGFWAHPPGAQAGTCVSQ 2 epitope Mouse TRPM4 QDRSGNCSMERGSWAHPEGPVAGSCVSQ 3 epitope Rat, mouse, ATCLQLAMQADARAFFAQDGVQSLLTQKWWG 4 human TRPM4 Forward primer 5′-CTGGTTCTCGCCTTCTTTTG-3′ 5 detection Reverse primer 5′-CATGAAGTCGATGCAGAGGA-3′ 6 detection siRNA sense 5′-CGCUAGUAGCAGCAAAUCUtt-3′ 7 siRNA antisense 5′-AGAUUUGCUGCUACUAGCGtq-3′ 8 Forward primer GCGAATTCCAGGACCGCAGTAGTAACTGCTC 9 cloning TGCCGAGCG Reverse primer CGGTCGACTCACTGGGACACACAGGAGCCTG 10 cloninq

A preferred embodiment of the invention relates to the antibody or fragment thereof being a polyclonal, monoclonal or humanized antibody.

Antibodies raised to this region of the TRPM4 protein according to the invention have been found to inhibit TRPM4 activity. The antibodies of the invention bind to and inhibit TRPM4 homomer channels, but may also be capable of inhibiting any channel formed by TRPM4.

More specifically, the antibody or fragment thereof is preferably raised using a TRPM4 peptide sequence selected from the group comprising or consisting of the rat peptide sequence SEQ ID NO: 1, the human peptide sequence SEQ ID NO: 2 and the mouse peptide sequence SEQ ID NO: 3, or an antigenic variant or fragment thereof. The term ‘variant’ has been defined above.

Preferably the peptide sequence used to raise the TRPM4 inhibitory antibody consists of the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; more preferably the peptide consists of SEQ ID NO: 1 or SEQ ID NO: 2. Antibodies raised to the rat peptide of SEQ ID NO: 1 bind to TRPM4 channels from rat, human and mouse species.

TRPM4 channel peptides from other species which are homologous to the peptide sequences of SEQ ID NOs: 1-3 may also be suitable as antigens for use according to the invention. Thus the scope of the invention is intended to encompass such homologous peptides.

It is important in the clinical setting that the antibody does not itself elicit an immune response in the subject. Therefore, it has become common practice to minimise or eliminate the immunogenicity of antibodies raised in other species used for human treatment by humanizing them.

Techniques have been developed for the production of humanized antibodies [See, e.g., Queen, U.S. Pat. No. 5,585,089 and Winter, U.S. Pat. No. 5,225,539, which are incorporated herein by reference in their entirety]. An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hyper variable regions, referred to as complementarity determining regions (CDRs). The extent of the framework region and CDRs have been precisely defined [see, “Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services (1983), incorporated herein by reference in their entirety]. Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

One type of antibody or fragment thereof suitable for human use is a mouse-human chimeric antibody.

Techniques developed for the production of “chimeric antibodies” [Morrison, et al., Proc Natl Acad Sci, 81: 6851-6855 (1984); Neuberger, et al., Nature 312: 604-608 (1984); Takeda, et al., Nature, 314: 452-454 (1985), incorporated herein by reference in their entirety] by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. For example, the genes from a mouse antibody molecule specific for a TRPM4 epitope can be spliced together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region [See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety].

Preferably, the mouse VH domain is ligated to human IgG1 CH domain and the mouse VL domain is ligated to human light chain kappa constant (CL) domain.

In one aspect of the invention, there is provided an antibody or a fragment thereof wherein said antibody or fragment inhibits TRPM4 currents. TRPM4 forms homomer channels and, as described herein, there is some evidence albeit conflicting that TRPM4 may also associate with SUR1 to form a channel. The data shown herein indicates the antibody of the invention binds to TRPM4 homomer channels.

In a preferred embodiment of the invention, there is provided an antibody or a fragment thereof for use in treating ischemic stroke. The antibody or fragment thereof binds to TRPM4 to inhibit TRPM4 activity. More specifically, the antibody or fragment thereof binds to a TRPM4 epitope selected from the group comprising, essentially consisting of, or consisting of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or a variant or fragment thereof.

In a preferred embodiment of the invention, there is provided a method of treating ischemic stroke, comprising administering to a subject in need thereof an efficacious amount of at least one TRPM4 inhibitor. Preferably the at least one inhibitor is an antibody or a fragment thereof which specifically binds to TRPM4, or is a TRPM4-specific siRNA.

The at least one TRPM4 inhibitor may be administered in combination with one or more thrombolytic agents. Thrombolytic agents are only used for ischemic stroke. The most commonly used drug for thrombolytic therapy is tissue plasminogen activator (tPA), but other drugs such as Lanoteplase, Reteplase, Staphylokinase, Streptokinase (SK), Tenecteplase and Urokinase can do the same thing. tPA is an enzyme found naturally in the body that converts, or activates, plasminogen to dissolve a blood clot. It is normally administered intra venously (IV) to the stroke patient during the acute phase and should be given within 3 to 4.5 hours of symptom onset.

In a preferred embodiment, the at least one TRPM4 inhibitor is administered during the acute stage and/or the chronic stage. More preferably, administration is during the acute stage.

From previous few studies on therapeutic antibodies for stroke, the dose of antibody for IV injection in the rat stroke model is about 200 μg. The IV dose of an antibody used in a failed human trail was 160 mg for a loading dose and subsequently 40 mg for maintenance for 4 days.

Preferably, the antibody is a polyclonal antibody, a mouse monoclonal antibody, or a humanized monoclonal antibody as described above, or a fragment thereof.

Various procedures known in the art may be used for the production of polyclonal antibodies to the polypeptide of the invention, or immunogenic fragment thereof. For the production of antibody, various host animals can be immunised by injecting the polypeptide or an immunogenic variant or fragment thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the peptide of the invention or variant or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). The peptide of the invention or immunogenic variant or fragment may be further combined with any adjuvant known in the art [for example, Hood et al., in Immunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park, Calif., 1984, herein incorporated by reference].

In particular, the polyclonal antibody may be produced by a method comprising the steps:

-   -   immunising an animal with a polypeptide consisting of the         sequence SEQ ID NO: 1, or an immunogenic variant or fragment         thereof;     -   isolating antibodies from said animal; and     -   screening the isolated antibodies with the polypeptide, thereby         identifying a polyclonal antibody that specifically binds to a         polypeptide comprising the sequence SEQ ID NO: 1.

An example of the method used for the production of an antibody of the present invention is given in Example 1, which provides a method used for the production of polyclonal antibody M4P using New Zealand white rabbits.

For the preparation of monoclonal antibodies directed towards the polypeptide of the invention or immunogenic variant or fragment thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include, but are not limited to, the hybridoma technique originally developed by Kohler et al., Nature, 256: 495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today, 4:72, (1983)], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985]. Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus, [e.g., Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; or U.S. Pat. No. 4,493,890].

In particular, the monoclonal antibody can be produced according to the method comprising the steps:

-   -   immunising an animal with a polypeptide consisting of the         sequence SEQ ID NO: 1, or an immunogenic variant or fragment         thereof;     -   isolating antibody-producing spleen cells and fusing them with         immortalised (myeloma) cells in the presence of PEG to form         hybridoma cells;     -   culturing the hybridoma cells and isolating clonal anti-TRPM4         monoclonal antibody producing cell lines; and     -   isolating from the clonal cell lines a monoclonal antibody that         specifically binds to the polypeptide sequence SEQ ID NO: 1 on         TRPM4.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting the binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting the binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of a polypeptide of the invention (for example, any one of SEQ ID NOS: 1 to 3), one may assay generated hybridomas for a product which binds to a polypeptide fragment containing such an epitope. For selection of an antibody specific to a polypeptide according to the invention from a particular species of animal, one can select on the basis of positive binding with the polypeptide of the invention expressed by or isolated from cells of that species of animal.

In a preferred embodiment of the invention there is provided a method of treating ischemic stroke with a siRNA, wherein the siRNA comprises or consists of a sense oligonucleotide SEQ ID NO: 7 and an antisense oligonucleotide SEQ ID NO: 8.

In a preferred embodiment of the invention, treatment with a TRPM4 inhibitor increases angiogenesis in the subject.

In another preferred embodiment of the invention, treatment with a TRPM4 inhibitor reduces infarct volume in the subject.

In a preferred embodiment of the invention, treatment with a TRPM4 inhibitor extends the therapeutic time window for reperfusion.

In another aspect of the invention there is provided the use of at least one TRPM4 inhibitor for the preparation of a medicament for the treatment of ischemic stroke. In a preferred embodiment, the at least one inhibitor is an antibody or a fragment thereof which specifically binds to the sequence which lies between S5 and the P-loop of TRPM4, or is a siRNA which specifically inhibits TRPM4 activity.

Preferably the antibody is a polyclonal antibody, a monoclonal antibody, or a humanized antibody or an antigen-binding fragment thereof. The monoclonal antibody may be a mouse monoclonal antibody, and the humanized antibody may be a humanized monoclonal antibody.

In a preferred embodiment, the siRNA comprises a sense oligonucleotide comprising or consisting of the sequence SEQ ID NO: 7 and an antisense oligonucleotide comprising or consisting of the sequence SEQ ID NO: 8 (Table 1). The siRNA may be synthesized by known methods or purchased, for example, from Ambion, Life Technologies Corporation, USA.

In yet another aspect of the present invention there is provided a pharmaceutical composition comprising a TRPM4 inhibitory antibody or fragment thereof according to any aspect of the present invention and, optionally a thrombolytic agent as hereinbefore described.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, adjuvant, diluent and/or detergent. Such formulations therefore include, in addition to the antibody, a physiologically acceptable carrier or diluent, possibly in admixture with one or more other agents such as other antibodies, thrombolytic agents or drugs, such as an antibiotic. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose and buffered saline. Alternatively, the antibody or fragment thereof may be lyophilized (freeze dried) and reconstituted for use when needed by the addition of an aqueous buffered solution as described above. Routes of administration are routinely intravenous.

Another aspect of the invention provides a process for the production of an antibody or fragment thereof according to any aspect of the invention, comprising administering to a mammal a TRPM4 peptide or antigenic variant or fragment thereof as hereinbefore described.

In a preferred embodiment, the peptide is selected from the group comprising or consisting of a peptide consisting of the amino acid sequence SEQ ID NO: 1, a peptide consisting of the amino acid sequence SEQ ID NO: 2 and a peptide consisting of the amino acid sequence SEQ ID NO: 3, or an antigenic variant or fragment thereof. Preferably the peptide consists of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

The peptides of the invention may be produced for immunization purposes synthetically or via expression constructs which encode them. An example of a suitable expression vector is the bacterial plasmid pGEX-4T-1 (Amersham Pharmacia Biotech, now GE Healthcare Life Sciences), which encodes a fusion protein with a thrombin cleavage site.

Suitable oligonucleotide primers for use in amplifying and cloning TRPM4 peptide into pGEX-4T-1 include: forward primer SEQ ID NO: 9 and reverse primer SEQ ID NO: 10 (Table 1).

Modifications and changes may be made in the structure of the DNA segments which encode the peptides and still obtain a functional molecule that encodes a peptide that can elicit an immune response. The nucleic acid molecules according to the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same peptide (for example, the peptides with SEQ ID NOs: 1-3). The nucleic acid molecules according to the invention may have sequence changes that cause a conservative amino acid substitution that does not significantly reduce the TRPM4 inhibitory activity of antibodies directed to such altered peptides. The isolated nucleic acid molecules according to the invention encompass segments that are not found as such in the natural state. Thus, the invention encompasses recombinant nucleic acid molecules incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).

The DNA may be expressed in a suitable host to produce a polypeptide comprising the TRPM4 peptide according to any aspect of the invention. Thus, the DNA encoding the peptide of the invention may be used in accordance with known techniques, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the peptide according to the invention.

In yet another aspect of the present invention there is provided at least one plasmid or vector comprising the nucleic acid molecule according to any aspect of the present invention.

In yet another aspect of the present invention there is provided a kit for treating stroke, the kit comprising at least one antibody or a fragment thereof according to any aspect of the invention and/or at least one siRNA according to any aspect of the invention and, optionally, at least one thrombolytic agent.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

Materials and Methods Animal Model

This study was approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Neuroscience Institute, Singapore. As per the approved protocol, male Wistar rats weighing approximately 300 g were subjected to permanent or transient middle cerebral artery occlusion (MCAO). Prior to surgery, the animals were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) intra-peritoneally. Under an operating microscope, the left common carotid artery was exposed and temporarily ligated using a vascular clip (Aesculap, B. Braun, Germany). Next, the left external carotid artery (ECA) and internal carotid artery (ICA) were dissected from the surrounding tissues. Occipital artery and superior thyroid artery (branches of ECA) were occluded. The ECA was then ligated with a 4-0 silk suture. The ICA was also free from the adjacent vagus nerve. Subsequently, a loose knot was made at the ECA stump near the bifurcation with a 4-0 silk suture. The extra cranial branch of ICA was temporarily ligated with a vascular clip as well. The distal end of the ECA was cut and a silicon coated filament (0.37 mm, Cat #403756PK10, Doccol Corp, Redlands, Calif.) was introduced into the ICA through the ECA stump. Subsequently, the suture around the ECA stump was tightened around the intraluminal filament, and the micro vascular clip was removed. The filament was then gently advanced from the ECA to the ICA lumen for approximately 18-20 mm. The ligation on the CCA was released after the suture on the ECA-intraluminal filament was tightened. The sham-operated rats underwent similar procedures, except for the insertion of the suture. For the transient MCAO model, the filament was removed at 2 hours or 5 hours after occlusion.

Infarct Volume Measurement

2,3,5-Triphenyltetrazolium chloride (TTC) staining relies on the ability of the dehydrogenase enzymes and cofactors present in the living tissue to react with tetrazolium salts, the main component of the TTC solution, to form a formazan pigment. After the animals were euthanized, the brains were collected, and the cerebellum and overlying membranes were removed. The brain was sectioned into 2-mm-thick coronal slices using a brain-sectioning block. The sections were stained with 0.1% TTC (Sigma, USA) solution at 37° C. for 30 minutes and then preserved in 4% formalin solution. The sections were scanned and the infarct size was analyzed using an image analyzer system (Scion image from Windows, Microsoft).

Immunofluorescent Staining

The animals were euthanized and perfused with saline followed by 4% paraformaldehyde (PFA). The brains were then collected and post-fixed with 4% PFA for 2 hours. Dehydration was subsequently carried out by immersing the brain in a 15% sucrose solution, followed by 30% sucrose solution. Next, the rat brain was cryosectioned at 20 μm of thickness. After washing with 0.2% Triton X-100 phosphate buffered saline (PBST), 100 μl of the blocking serum (10% goat serum and 1% bovine serum albumin in 0.2% PBST) was added to the sections for 1 hour. The brain sections were then incubated with primary antibodies overnight at 4° C. The primary antibodies used in the study are: anti-TRPM4 (sc-27540, Santa Cruz, Calif., USA), anti-NeuN (MAB377, Millipore), anti-GFAP (IF03L, Calbiochem, Millipore), anti-smooth muscle actin (CBL171, Millipore), and anti-vWF (AB7356, Millipore, Mass., USA). On the following day, the tissue sections were washed 3 times with TNT wash buffer (0.1 M tris-HCl buffer pH7.5 containing 0.15 M NaCl and 0.05% Tween 20). The slides were incubated with secondary antibodies for 1 hour at room temperature. After washing 3 times of wash buffer, the slides were mounted with FluorSave™ reagent (Merck, Germany). The results were visualized by a laser scanning confocal microscope (Fluoview BX61, Olympus). The negative control underwent an identical procedure with the exception of the primary antibody incubation; no positive signal was observed.

Capillary Counting

Capillary counting was conducted stereologically. Every fifth 100-μm brain section across the entire region of infarction was counted. For random sampling, six fields per brain section were randomly chosen under a confocal microscope under 40-× magnification. The number of blood vessels was measured by counting the number of elongated tube-like structures with positive vWF immunoreactivity.

Western Blot

Tissues from the penumbra region and the contralateral control sites were collected from the TTC-stained brain slices as described earlier [Zhao H, et al., J Neurosci, 25, 9794-806 (2005)], incorporated herein by reference. The brain tissues were homogenized in 300 μl of HEPES lysis buffer (20 mM HEPES, 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA) with freshly added protease inhibitors (1:50 dilution, Roche Diagnostics). The homogenized samples were then centrifuged at 14000 rpm for 15 minutes, and the supernatants were collected. Protein concentration was determined using the Bradford assay. For western blot analysis, 200 μg of lysate (with 2× protein loading dye) was separated on an 8% SDS-PAGE gel. The gel was then transferred overnight at 30 V onto a PVDF membrane at 4° C. Subsequently, the membrane was blocked with 1% BSA in 1×PBS+0.1% Tween 20 for 1 hour and probed with an anti-TRPM4 goat polyclonal antibody (1:2000 dilution, SC27540, Santa Cruz Biotechnology) overnight at 4° C. The next day, the membrane was washed 3 times with 0.1% Tween 20 in 1×PBS for 10 minutes. The membrane was then probed with a goat secondary antibody conjugated to HRP (1:5000 dilution, A5420, Sigma Aldrich) for 1 hour. After washing, the TRPM4 band was detected using the Amersham ECL Western Blotting Analysis System (RPN2109, GE Healthcare).

Human Umbilical Vein Endothelial Cells (HUVECs) Culture and Hypoxia Induction

HUVECs (Lonza, Wokingham, UK) were cultured at 37° C. with 5% CO₂. The culture medium is endothelial growth medium-2 (EGM-2) consisting of endothelial basal medium (EBM), supplemented with 2% fetal bovine serum, hydrocortisone, hFGF, VEGF, R3-IGF-1, ascorbic acid, HEGF, GA-1000, and heparin (Lonza, Wokingham, UK). Cells at passage 6-10 were used for experiments. HUVECs were subjected to oxygen/glucose deprivation (OGD). To achieve supplement deprivation, EGM-2 was changed to EBM without fetal bovine serum or growth supplements. Hypoxia was induced by culturing the cells in a hypoxic chamber (Stem Cell Technologies, Vancouver, Canada) with 1% O₂ and 5% CO₂ at 37° C. for 24 hours.

HEK Cell Transfection with Mouse TRPM4 and Prolonged Exposure to M4P

HEK293 cells were transiently transfected with mouse TRPM4 using lipofectamine 2000 (lifetechnologies). Positive cells are in green color labeled with GFP. M4P antibody was applied into the culture medium and harvested at different time points (15 min-2 days) for in vitro studies on the binding of M4P to TRPM4 channel on cell membrane. Control rabbit IgG was used as a control. Immunofluorescent staining was used to study the surface binding of M4P. Primary antibody was omitted, while goat anti-rabbit secondary antibody was applied for staining. For hypoxia treatment, M4P or control IgG was added into the culture medium and the cells were incubated under Oxygen-Glucose deprivation for 12 hr-2 days. The cell survival was measured with Trypan blue and MTT assay.

TRPM4 Channel Currents in Mouse TRPM4 Transfected HEK Cells Exposed to M4P

Cells were transfected as described above. Whole-cell currents were recorded under voltage clamp with Axopatch 200B amplifier (Molecular Devices Corp. Sunnyvale, Calif., USA). Data were digitized at 10 kHz and filtered at 1 kHz. The pClamp 10.2 software was used for data acquisition and analysis. The internal pipette solution contained (in millimole/liter): CsCl 140, NaCl 5, MgCl2 1, BAPTA 1, CaCl₂) 0.83, and pH 7.2 adjusted with CsOH. The bath solution contained (in millimole/liter): NaCl 140, CsCl 5, CaCl₂) 2, MgCl2 1, glucose 10, HEPES 10, and pH 7.4 adjusted with NaOH.

Trypan Blue Exclusion and MTT Assay

Cell viability was determined using the trypan blue exclusion method. The cells were trypsinised and incubated with trypan blue. The number of cells was quantified by light microscopy using a haemocytometer chamber and is expressed as the percentage of viable cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Roche, Switzerland) was performed to assess cell viability. Cell viability was quantified by the amount of MTT reduction. The cells were incubated with anhydrous MTT for 4 hours, and the product was solubilized in a DMSO solution. The optical density was measured at 540 nm. The data is expressed as the percentage of viable cells.

Tube Formation

HUVECs were divided into the following groups: Normoxia; Normoxia+5 μM 9-phenanthrol (Sigma); Hypoxia; and Hypoxia+5 μM 9-phenanthrol. A 24-well culture plate was precoated with 250 μl of growth factor-reduced Matrigel (Sigma, USA) at 37° C. for 30 minutes. 24 hours after OGD, 4×10⁴ cells (in 300 μl of EGM-2) from each group were seeded onto the Matrigel-coated plates. The formation of capillary structures was examined under a light microscope after 4 hours. This study was replicated more than 4 times.

Reverse Transcriptase PCR

Total RNA was extracted using the TRIzol reagent (Invitrogen, Life Technologies Corporation, USA) according to the manufacturer's protocol. Superscript III (Invitrogen, Life Technologies Corporation, USA) was used to generate first strand cDNA. The PCR was performed as follows: a denaturation step at 95° C. for 5 min; 35 cycles of 94° C. for 30 seconds, 60° C. for 45 seconds, and 72° C. for 30 seconds; and a final extension step at 72° C. for 10 min. GAPDH was used as the endogenous control. The primers used for TRPM4 were as follows: 5′-CTGGTTCTCGCCTTCTTTTG-3′ (forward; SEQ ID NO: 5; Table 1) and 5′-CATGAAGTCGATGCAGAGGA-3′ (reverse; SEQ ID NO: 6; Table 1); and GAPDH: 5′-GAAGGTGAAGGTCGGAGTCAACG-3′ (forward; SEQ ID NO: 14) and 5′-TGCCATGGGTGGAATCATATTGG-3′ (reverse; SEQ ID NO: 15).

In Vivo siRNA Delivery

TRPM4 in vivo Ready siRNA was purchased from Ambion, Life Technologies Corporation, USA. The sequences were as follows: sense 5′-CGCUAGUAGCAGCAAAUCUtt-3′ (SEQ ID NO: 7) and antisense 5′-AGAUUUGCUGCUACUAGCGtg-3′ (SEQ ID NO: 8). Immediately after operation, the rats received a loading dose of 19 nmole siRNA intravenously. Subsequently, 6 nmole siRNA was given via the jugular vein through an implanted mini-osmotic pump (Alzet, Durect Corporation). The infusion rate was 7.8 μl/hour for 24 hours.

Behavioral Analysis

Motor function after MCAO was evaluated using a rotarod apparatus (Ugo Basile, Italy). The performance of the rats was measured by observing the latency with which the rats fell off the rotarod. Three days before the surgery, the rats received 3 training trials each day with 15-minute intervals. The accelerating rotarod was set from 4 to 80 rpm within 10 minutes. The mean duration of time that the animals remained on the device was recorded 1 day before MCAO as an internal baseline control. At different time points following surgery, the mean duration of latency was recorded and compared to the internal baseline control.

Antibody Production

The procedure for polyclonal antibody production was described before [Liao P, et al., J Biol Chem 279: 50329-35 (2004)]. In brief, part of the extracellular domain of rat TRPM4 (FIG. 1; SEQ ID NO: 11), consisting of the amino acid sequence QDRSSNCSAERGSWAHPEGPVAGSCVSQ (SEQ ID NO: 1; Table 1), corresponding to amino acids 949-952 and 985-1008 of the mature protein (FIG. 1; SEQ ID NO: 11; NCBI Accession# NP_001129701 XP_574447) was cloned in frame into the plasmid expression vector pGEX-4T-1. The primers used for cloning were: Forward primer: GCGAATTCCAGGACCGCAGTAGTAACTGCTCTGCCGAGCG (SEQ ID NO: 9; Table 1) and Reverse primer: CGGTCGACTCACTGGGACACACAGGAGCCTG (SEQ ID NO: 10; Table 1). Part of the intracellular domain before the first transmembrane segment of rat TRPM4, corresponding to the amino acid sequence ATCLQLAMQADARAFFAQDGVQSLLTQKWWG (SEQ ID NO: 4) and amino acids 650-680 of the mature protein (FIG. 1; SEQ ID NO: 11) and identical to the sequences in human and mouse TRPM4 (FIGS. 2-3; SEQ ID NOs: 12-13), was also used to generate antibodies. GST-fused protein was extracted from bacteria and purified with glutathione-agarose (Sigma). Purified protein was used to immunize female New Zealand White rabbit once a month. Complete Freund's adjuvant (Sigma) was first injected for immunization, and incomplete Freund's adjuvant was used in subsequent injections once a month. Serum collected from rabbits was pre-absorbed with GST protein to remove non-specific antibodies, and polyclonal antibody was affinity-purified from immobilized TRPM4 protein with an IgG elution buffer (Pierce). The eluted antibody concentration was 1 μg/μl and the ‘extracellular domain’ antibody was named M4PAb. Serum from rabbits before immunization was used as pre-immune control.

Epitope for Humanized Antibody Production

The epitope from rat TRPM4 targeted by M4P antibody lies between S5 and the P-loop of the TRPM4 channel and corresponds to amino acids 949-952 and 985-1008 of SEQ ID NO: 11 (shown schematically in FIG. 4).

Antibody M4P was found to also bind to, and inhibit, TRPM4 on human cells. The corresponding amino acid sequence of the human TRPM4 channel is RDSDSNCSSEPGFWAHPPGAQAGTCVSQ (SEQ ID NO: 2) corresponding to amino acids 955-958 and 991-1014 of the mature isoform 1 of TRPM4b (FIG. 2; SEQ ID NO: 12).

Humanized Antibody Production Against TRPM4 Channel

Methods for generating rabbit and mouse monoclonal antibodies are known, as are methods for humanizing them [Cianfriglia M, et al., Hybridoma; 2(4): 451-7 (1983); Riechmann L, et al., Nature 332(6162): 323-7 (1988); Verhoeyen M, et al., Science 239(4847):1534-6 (1988), incorporated herein by reference].

Stacie I: Antibody Sequencing

Based on the study on rat TRPM4 channel with polyclonal antibody M4P, the amino acid sequence of human TRPM4 suitable for generating humanized antibody is RDSDSNCSSEPGFWAHPPGAQAGTCVSQ (SEQ ID NO: 2). This polypeptide is produced and injected into BALB/c mice as the antigen over 3-4 weeks. The sera is then evaluated for anti-TRPM4 responses by analyzing protein-protein interaction in, for example an ELISA assay. Following that, antibody-producing murine splenocytes are fused with myeloma cells and clonal anti-TRPM4 antibody producing hybridoma cell lines are selected.

The mouse monoclonal antibody against human TRPM4 can be characterized by western blot, in vitro binding assay, electrophysiological study, and therapeutic efficacy on cultured cells as well as in rodent stroke models. Western blot will be carried out using cell lines expressing human TRPM4 and samples from human stroke patients. A binding assay can be carried out by incubating cultured TRPM4 expressing cells with the mouse monoclonal antibody. The surface binding is studied by immunostaining. Additionally, the antibody is injected into MCAO rats to determine whether the antibody binds to the endothelium following stroke. Standard Trypan blue exclusion and MTT assays can then be used to evaluate whether the mouse monoclonal antibody can protect cultured endothelial cells from hypoxic treatment. The therapeutic efficacy is evaluated by measuring the tissue loss following the administration of the therapeutic monoclonal antibody. Further characterization of the antibody involves using patch clamp methods, as described in [Nilius B, et al., J Biol Chem, 280(24): 22899-906 (2005)] and herein incorporated by reference, to determine whether the antibody can block TRPM4 currents.

Stage II: Antibody Humanization

Bioinformatics (sequence analysis and modeling) software is used to blend the antibody variable regions from stage I to human donor sequences, creating a panel of humanized heavy and light chains. The humanized chains are then codon optimized for expression in a mammalian system and synthesized (Assay Biotechnology, CA, US). Selections of human constant domain sequences are available and the isotype, class, and subclass of the final antibody can also be specified at this stage.

Stage III: Small Scale Transient Transfection

Each humanized antibody is cloned into a proprietary expression vector for small scale transient transfection using known methods [for example, Karagiannis P, et al., Cancer Immunol Immunother 58:915-30 (2009)], incorporated herein by reference. Each construct will undergo small scale expression and purification; the resulting antibodies are tested for affinity to the target as described above.

Stage IV: Stable Cell Line Development

The selected humanized antibody is then used to generate a stable cell line using a mammalian gene expression system including dihydrofolatereductase or glutamine synthetase amplification systems and the ubiquitous chromatin opening element technologies as described, for example, by Chandrashekran A, et al., [FEBS Open Bio 4: 266-75 (2014)], incorporated herein by reference. Once satisfied with the antibody characteristics the cell line can be further optimized for high yield expression and validated for scale-up.

Statistics

All of the results are presented as the mean±S.E.M. Data were graphed using Prism, version 4 (GraphPad Software, CA). Student's t test was used to compare two sample means and one way ANOVA followed by Dunnett's post hoc tests was used to compare the means of data from three or more groups. The results were considered significant if P<0.05.

Results

Upregulation of TRPM4 in the Vascular Endothelium after MCAO

To investigate the role of TRPM4 in ischemic stroke, the middle cerebral artery in rats was permanently occluded. The infarction was located in ipsilateral cortex and striatum (left hemisphere in this study). DAB staining of the capillaries with the endothelial marker von Willebrand factor (vWF) in the contralateral and ipsilateral regions indicated prominent angiogenesis in the penumbra region 1 day post stroke (FIG. 5A). Endothelial vWF staining was stronger in the penumbra region than in the contralateral brain tissues possibly due to endothelial activation and/or dysfunction after stroke [De Meyer S F, et al., Stroke, 43, 599-606 (2012)].

Using a TRPM4-specific antibody, strong staining of TRPM4 was detected in the penumbra region that was co-labeled with vWF 1 day post MCAO (FIG. 5B). TRPM4 was almost undetectable in the vascular endothelium in the uninjured contralateral hemisphere (FIG. 5B) and sham-operated brain (FIG. 6A). Another TRPM channel, TRPM5, which is structurally similar to TRPM4, was not expressed in the endothelium (FIG. 6B).

Western blot analysis revealed a two-fold upregulation of TRPM4 in the penumbra region (FIG. 4a ) as compared to the contralateral region. The rat TRPM4 is approximately 135 kDa, suggesting that this protein represents the longer TRPM4b isoform [Nilius B, et al., J Biol Chem, 278, 30813-20 (2003)]. The same antibody was able to detect the mouse TRPM4 protein which was expressed in HEK cells as a positive control, confirming the specificity of the antibody for TRPM4. We continuously observed the size of rat TRPM4 was slightly smaller than the mouse TRPM4.

The expression of TRPM4 was further studied in the vascular endothelium within the penumbra at the following time points post MCAO: 0 hour, 6 hour, 1 day, 3 days, and 7 days (FIG. 7). Strong TRPM4 staining was observed in the endothelium as early as 6 hours. The expression levels remained prominent at 1 day post operation. The almost complete colocalization of TRPM4 with vWF indicated that the majority of the endothelial cells within the penumbra region expressed high levels of TRPM4 following stroke. However, by day 3, TRPM4 expression began to decrease gradually. Many vWF-positive cells did not express TRPM4. Furthermore, vascular fragmentation was observed within penumbra region after stroke induction, a typical sign of the loss of vascular integrity. This fragmentation was prominent by day 3 and correlated with the reduced expression of TRPM4. Thus, TRPM4 upregulation in the vascular endothelium may contribute to capillary death post stroke.

In Vitro Blockade of TRPM4 in Human Umbilical Vein Endothelial Cells (HUVECs) Induces Tube Formation

This hypothesis was tested in HUVECs under oxygen/glucose deprivation (OGD). The cells were exposed to hypoxic conditions and starved of glucose and serum/growth factors. After 24 hours of OGD, significant cell death occurred. A two-fold increase of TRPM4 mRNA by RT-PCR (FIG. 8A) and a 1.7-fold increase of TRPM4 protein by Western blot (FIG. 8B) were observed after OGD treatment. Such increase was further confirmed by immunofluorescence staining (FIG. 8C).

To test the functional impact of TRPM4 on HUVECs, the cells were incubated with the TRPM4-specific blocker 9-phenanthrol. This blocker does not affect the TRPM5 channel [Grand T, et al., Br J Pharmacol, 153, 1697-705 (2008)], another TRPM channel with similar electrophysiological properties to TRPM4 [Vennekens R, Nilius B. Handb Exp Pharmacol, 269-85 (2007b); Nilius B, et al., Physiol Rev, 87, 165-217 (2007)]. A study was performed in which 9-phenantrol with the concentration ranged from 0.1 μM to 30 μM was added to HUVECs. No difference was observed in cell death between 0.1 μM and 5 μM. Cell death became prominent when 10 μM 9-phenanthrol was added to the culture medium. Very few cells survived treatment with 30 μM 9-phenanthrol (FIG. 9A). Thus, 5 μM 9-phenanthrol was used to treat HUVECs. Unexpectedly, 9-phenanthrol treatment did not reduce cell death after OGD (FIGS. 9B and C). However, a large enhancement of tube formation was observed on Matrigel by 9-phenanthrol treatment after OGD (FIG. 8D). This in vitro study shows that blocking TRPM4 improves endothelial functions under hypoxic conditions.

In Vivo TRPM4 Knockdown Enhances Angiogenesis and Reduces Infarction after MCAO

To demonstrate that TRPM4 is critical for vascular endothelial functions in vivo, a siRNA was used against rat TRPM4 in the animal model of MCAO. A total of 25 nmoles of siRNA was delivered into rats with a body weight of 300 g. A single dose of 19 nmoles was injected intravenously post operation and the remaining 6 nmoles was delivered into the jugular vein with an osmotic pump for 24 hours. Intravenous delivery reduces the loss of siRNA during absorption via other routes and maximizes contact with the endothelium.

Western blot analysis revealed that in the saline-treated animals, there was an upregulation of TRPM4 in the ipsilateral region and very low expression of TRPM4 in the contralateral region. Knockdown of TRPM4 via siRNA successfully prevented the upregulation of TRPM4 in the ipsilateral hemisphere 1 day post MCAO (FIG. 10A). TRPM4 protein levels were reduced to a level similar to that in the contralateral region. De novo expression of TRPM4 was previously shown to contribute to the damage of capillary integrity following SCI [Gerzanich V, et al., Nat Med, 15, 185-91 (2009)]. The capillary structure was studied in the rat brains 3 days post MCAO. In the saline-treated animals, the capillaries were generally short, and fragmentation was present in almost all capillaries (FIG. 10B). The capillaries within the penumbra region of the siRNA-treated animals were elongated without segmentation, indicating greater structural integrity. In addition, the number of capillaries in the penumbra region was increased 2.5-fold relative to the saline-treated animals (saline 8.8±0.8 vs. siRNA 21.4±0.5, P<0.0001, Student's t test). Thus, TRPM4 inhibition can promote angiogenesis in a rat model of stroke.

Next, it was examined whether TRPM4 deletion affects brain tissue loss after stroke. In the saline-treated animals 1 day post MCAO, large infarction was observed in the ipsilateral hemisphere according to TTC staining (FIG. 100). Both the cortex and striatum were affected. By contrast, siRNA treatment greatly reduced infarction. The cortex was preserved almost completely, while a smaller infarction occurred mainly in the striatum. The infarction volume was greatly reduced from 24±3.2% to 8.8±1% (P=0.0017, Student's t test) (FIG. 10C).

Utilising the transient MCAO model also indicated that TRPM4 inhibition by siRNA is beneficial for reducing infarction volume and improving functional recovery (FIG. 11). Moreover, siRNA treatment enhances angiogenesis, as seen by significantly increased diameters and lengths of cerebral blood vessels compared to treatment with scrambled siRNA treatment (FIG. 12).

In Vivo TRPM4 Knockdown Improves Motor Function after MCAO

A rotarod test was used to evaluate the motor function of the rats receiving siRNA treatment. The sham-operated animals performed well on the test (FIG. 13A). The performance of MCAO rats (n=8) receiving saline treatment decreased to 28±4% 1 day post operation and gradually improved in subsequent days. The performance of the 21 siRNA-treated MCAO rats greatly improved to 57±3.7% 1 day post MCAO, which is significantly higher than the saline group but lower than the sham-operated group (P<0.0001, one-way ANOVA followed by Dunnett's post hoc analysis). The effects of siRNA were most prominent 3 days post MCAO. There was no difference between the siRNA-treated animals and sham-operated animals (FIG. 13B). The protective effect lasted until day 5 post operation. By day 7, the motor function deficit in the siRNA-treated animals decreased to levels that were similar to those in the saline-treated animals (FIG. 13A).

Transient Protective Effect of TRPM4 Knockdown

The results from the rotarod test suggest that TRPM4 knockdown does not maintain functional improvement after the acute phase of stroke. To study the effects of TRPM4 knockdown on tissue loss after the acute phase, TTC staining was performed to measure the infarction volume at different time points (FIG. 14A). In the saline-treated rats, the infarct volume decreased gradually by day 7, indicating that the process of reabsorption was occurring. By contrast, the protective effects of siRNA treatment disappeared by day 5. Although the mean infarct volumes were smaller than those in the saline-treated animals, there was no difference between the two groups (FIG. 14B). Thus, the protective effect of TRPM4 knockdown appears to be transient and occurs during the acute phase of stroke. These results support that brain tissue loss correlates with functional changes after MCAO.

Antibody Binds to TRPM4 Channel in Live Cells and Reduces Cell Death after OGD

As no potent TRPM4 blockers can be used in vivo, an antibody was developed against TRPM4 that could potentially block the channel. After targeting various extracellular regions of TRPM4, one antibody M4P was able to bind to the TRPM4 channels in vivo (FIG. 15A). Incubation with M4P in live HEK cells transfected with TRPM4 showed a strong membrane localization, which was absent in control rabbit IgG treatment. To test the therapeutic effect, TRPM4 transfected HEK cells were incubated in hypoxic gas and in medium without glucose. Cell death was greatly reduced with M4P treatment after 12 hour OGD (FIG. 15B). In chemicals treated cells (Sodium Azide and 2-Deoxy-D-glucose), M4P yielded similar protective effect (FIG. 15C).

After 24 hours OGD M4P treatment increased cell survival both in HEK cells (FIG. 16A) and in human cerebral vascular endothelial cells (FIG. 16B). Although rats and humans share 61% identity in the epitope recognized by M4P, the M4P antibody successfully protected the human cerebral vascular endothelial cells from hypoxia.

The antibody directed to the intracellular region (SEQ ID NO: 4) of TRPM4 did not effectively bind to the TRPM4 channel in both transfected cells and human brain endothelial cells (data not shown).

A review of the progress of the development of therapeutic antibodies for stroke [Yu C Y, et al., Transl Stroke Res 4(5): 477-83 (2013)], suggests very few channels have been targeted. Antibodies against the NMDA receptor have been extensively studied, however, severe side effects led to the termination of clinical trials. As the NMDA receptor is ubiquitously expressed in the brain, blocking NMDA receptor after stroke could affect neurons within the healthy brain tissue and cause unwanted side effects. In contrast to the NMDA receptor, TRPM4 may be an ideal target as it is not abundantly expressed in healthy brain tissue and are therefore less likely to be affected by TRPM4 blockers. TRPM4 knockout mice appear healthy with no major deficits [Vennekens R, et al., Nat Immunol 8(3): 312-20 (2007a)] indicating that the side effects of blocking TRPM4 in other organs and tissues could be minimal during the time for stroke treatment. As a polyclonal antibody, M4P cannot be used directly in human patients. Therefore, a humanized antibody directed to the same region of TRPM4 that the M4P antibody binds to would be useful for treatment in human patients.

Antibody Binds to TRPM4 in Live Cells and is Internalized Under Prolonged Incubation

The in vitro study shows that antibody M4P could bind to the membrane TRPM4 channels after incubation for only 30 minutes and, under prolonged incubation, TRPM4 channels on the cell membrane can be internalized by M4P into the cytoplasm (FIG. 17).

Antibody Binds to TRPM4 Transfected HEK Cells and Inhibits Channel Currents.

Voltage clamp recordings showed that binding of antibody M4P to cells transfected with the rat TRPM4 channel inhibited whole-cell currents (FIG. 18).

Therefore, antibodies directed to the same epitope as M4P are likely to inhibit TRPM4 in two mechanisms: by blocking TRPM4 channel in the acute stage and by downregulating surface TRPM4 expression via internalizing membrane TRPM4 protein in the chronic stage.

Discussion

The ectopic expression and activation of TRPM4 is generally harmful to cells. Under pathological conditions, an increase in intracellular Ca²⁺ concentrations and the depletion of ATP leads to TRPM4 activation [Vennekens R, Nilius B., Handb Exp Pharmacol, 269-85 (2007b)], resulting in oncotic cell death due to unchecked Na⁺ influx [Gerzanich V, et al., Nat Med, 15, 185-91 (2009)]. The cessation of the blood supply to part of the brain after ischemic stroke can increase intracellular Ca²⁺ levels and lower ATP concentrations within the affected area; both of these events can enhance TRPM4 activities.

In the central nervous system, de novo expression of TRPM4 has also been identified in the capillaries following SCI [Gerzanich V, et al., Nat Med, 15, 185-91 (2009)]. In the present study a similar upregulation of TRPM4 protein was observed in the capillary endothelia after stroke. In cultured HUVECs, TRPM4 was expressed at basal levels under normoxic conditions, similar to a previous report [Becerra A, et al., Cardiovasc Res, 91, 677-84 (2011)]. In this study, TRPM4 expression is low in the healthy rat brain. The culture conditions may have caused the increased TRPM4 expression in HUVECs. OGD treatment increased TRPM4 expression at both the transcriptional and translational levels. Blocking TRPM4 channels with 9-phenanthrol enhanced tube formation, a sign of angiogenesis after hypoxia. Thus, the upregulation of TRPM4 is harmful to endothelial cells, and TRPM4 inhibition can protect HUVECs from hypoxic insult. This is supported by another study indicating that blocking TRPM4 can prevent HUVECs from lipopolysaccharide-induced cell death [Becerra A, et al., Cardiovasc Res, 91, 677-84 (2011)].

In SCI, de novo expression of TRPM4 in the endothelium caused capillary fragmentation, and deletion of TRPM4 greatly enhanced recovery by promoting angiogenesis [Gerzanich V, et al., Nat Med, 15, 185-91 (2009)]. The loss of vascular integrity was also apparent within the penumbra region using MCAO model in this study. TRPM4 knockdown via siRNA greatly promoted angiogenesis and improved the motor functions of the rats. This indicates that the upregulation of TRPM4 in the endothelium following stroke plays a similar pathological role as in traumatic SCI. The increased expression of TRPM4 in the capillaries after SCI led to secondary hemorrhage. It is possible that the upregulation of TRPM4 is a cause of hemorrhage transformation observed in many patients with ischemic stroke.

In the MCAO model, the animals that were treated with the siRNA displayed not only intact capillaries but also an increased number of capillaries compared to the saline-treated rats. However, blocking TRPM4 in HUVECs only improved tube formation without increasing the cell number. This could be due to the differences in the levels of growth factors in the two studies. In the animal model, VEGF and other growth factors generated after the onset of stroke could promote capillary proliferation, whereas in HUVECs under OGD, the serum and growth factors were completely removed. Thus, no cell proliferation was observed.

The ectopic expression of TRPM4 within the axonal processes contributes to the tissue damage induced by EAE [Schattling B, et al., Nat Med, 18, 1805-11 (2012)]. TRPM4 was also found in cerebral vascular smooth muscle cells. Blocking TRPM4 with antisense oligonucleotides greatly reduced pial artery constriction [Reading S A, Brayden J E, Stroke, 38, 2322-8 (2007)]. More experiments are needed to clarify the role of TRPM4 in neurons and vascular smooth muscles after stroke.

The expression of TRPM4 was transient after ischemic stroke. It peaked within 1 day and then gradually decreased. As angiogenesis occurs soon after the onset of stroke, TRPM4 is likely to affect the regeneration of capillaries. In fact, TRPM4 knockdown immediately after MCAO preserved vascular integrity, enhanced angiogenesis, and as a result, reduced infarction, and promoted functional recovery. However, the effect of siRNA treatment was transient. By day 5, the rotarod performances of the animals were similar to those of the saline-treated rats. These results suggest that TRPM4 upregulation only participates in endothelial damage during the acute phase of stroke. This is supported by the observation that by day 7, many vascular endothelial cells did not express TRPM4 channels. The mechanism for this unique expression pattern is not known. Acute stroke reperfusion treatments are currently restricted by the very narrow time window (<4.5 hours for intravenous thrombolysis), limiting this treatment to only a minority of patients. This is due to the progression of the ischemic penumbra to infarction over the first few hours following cerebral arterial occlusion [Astrup J, et al., Stroke, 12, 723-5 (1981)]. According to the animal data in the present study, blocking TRPM4 during the acute phase protects the brain tissue for as long as 5 days. Thus, blocking TRPM4 could potentially extend the therapeutic time window of acute reperfusion treatments.

Currently, there are no safe and specific TRPM4 blockers that can be used in vivo. A polyclonal antibody was developed, namely M4P, which can bind to and block TRPM4 channels. The data shows that M4P can bind to live cells expressing TRPM4 channels on the membrane surface. In cultured cells, M4P treatment significantly reduced hypoxia induced cell death and inhibited TRPM4 channel currents. In general, antibodies are large molecules and have difficulties in passing through the blood-brain barrier (BBB). However, the BBB integrity is compromised after stroke. Thus, antibodies can enter the brain areas that are affected by stroke. Furthermore, the main target of the M4P antibody is the endothelial TRPM4. It is easier for the antibody to bind to the TRPM4 channels on the capillaries. From the functional study, blocking TRPM4 during acute phase of stroke could extend the tissue loss to a later time point. Currently, the narrow time window of reperfusion therapy limits this therapy only to a small number of stroke patients. Blocking TRPM4 with antibodies could extend the time window and benefit more stroke patients.

In summary, the transient expression of TRPM4 during the acute phase following ischemic stroke is critical for capillary integrity. Blocking TRPM4 can protect brain tissue by promoting angiogenesis and represents a potential drug target for stroke therapy during both the acute and chronic stages.

As used herein, the term ‘comprising’ does not preclude the presence of additional steps or substances in the methods and compositions, respectively, of the invention, and is understood to include within its scope the more restrictive terms ‘consisting of’ and ‘consisting essentially of’ features defined in the claimed invention.

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1.-7. (canceled)
 8. A method of treating ischemic stroke, comprising administering to a subject in need thereof an efficacious amount of at least one TRPM4 inhibitor.
 9. The method according to claim 8, wherein the at least one inhibitor is an antibody or a fragment thereof which specifically binds to TRPM4, or is a TRPM4-specific siRNA.
 10. The method according to claim 9, wherein the antibody is a polyclonal antibody, a mouse monoclonal antibody, or a humanized monoclonal antibody, or a fragment thereof.
 11. The method according to claim 9, wherein the siRNA comprises a sense oligonucleotide comprising SEQ ID NO: 7 and an antisense oligonucleotide comprising SEQ ID NO:
 8. 12. The method according to claim 8, wherein the at least one TRPM4 inhibitor is administered in combination with one or more thrombolytic agents.
 13. The method according to claim 8, wherein the at least one TRPM4 inhibitor is administered during the acute stage and/or the chronic stage.
 14. The method according to claim 8, wherein treatment increases angiogenesis in the subject.
 15. The method according to claim 8, wherein treatment reduces infarct volume in the subject.
 16. The method according to claim 8, wherein treatment extends the therapeutic time window for reperfusion. 17.-25. (canceled) 